All forms of life must duplicate the genetic material to propagate the species. The process by which the DNA in a chromosome is duplicated is called replication. The replication process is performed by numerous proteins that coordinate their actions to duplicate the DNA smoothly. The main protein actors are as follows (reviewed in Kornberg et al., DNA Replication, Second Edition, New York: W.H. Freeman and Company, pp. 165-194 (1992)). A helicase uses the energy of ATP hydrolysis to unwind the two DNA strands of the double helix. Two copies of the DNA polymerase use each “daughter” strand as a template to convert them into two new duplexes. The DNA polymerase acts by polymerizing the four monomer unit building blocks of DNA (the 4 dNTPs, or deoxynucleoside triphosphates are: dATP, dCTP, dGTP, dTTP). The polymerase rides along one strand of DNA using it as a template that dictates the sequence in which the monomer blocks are to be polymerized. Sometimes the DNA polymerase makes a mistake and includes an incorrect nucleotide (e.g., A instead of G). A proofreading exonuclease examines the polymer as it is made and excises building blocks that have been improperly inserted in the polymer.
Duplex DNA is composed of two strands that are oriented antiparallel to one another, one being oriented 3′-5′ and the other 5′ to 3′. As the helicase unwinds the duplex, the DNA polymerase moves continuously forward with the helicase on one strand (called the leading strand). However, due to the fact that DNA polymerases can only extend the DNA forward from a 3′ terminus, the polymerase on the other strand extends DNA in the opposite direction of DNA unwinding (called the lagging strand). This necessitates a discontinuous ratcheting motion on the lagging strand in which the DNA is made as a series of Okazaki fragments. DNA polymerases cannot initiate DNA synthesis de novo, but require a primed site (i.e., a short duplex region). This job is fulfilled by primase, a specialized RNA polymerase, that synthesizes short RNA primers on the lagging strand. The primed sites are extended by DNA polymerase. A single-stranded DNA binding protein (“SSB”) is also needed; it operates on the lagging strand. The function of SSB is to coat single stranded DNA (“ssDNA”), thereby melting short hairpin duplexes that would otherwise impede DNA synthesis by DNA polymerase.
The replication process is best understood for the Gram negative bacterium Escherichia coli and its bacteriophages T4 and T7 (reviewed in Kelman et al., “DNA Polymerase III Holoenzyme: Structure and Function of Chromosomal Replicating Machine,” Annu. Rev. Biochem., 64:171-200 (1995); Marians, K. J., “Prokaryotic DNA Replication,” Annu. Rev. Biochem., 61:673-719 (1992); McHenry, C. S., “DNA Polymerase III Holoenzyme: Components, Structure, and Mechanism of a True Replicative Complex,” J. Bio. Chem., 266:19127-19130 (1991); Young et al., “Structure and Function of the Bacteriophage T4 DNA Polymerase Holoenzyme,” Am. Chem. Soc., 31:8675-8690 (1992)). The eukaryotic systems of yeast (Saccharomyces cerevisae) (Morrison et al., “A Third Essential DNA Polymerase in S. cerevisiae,” Cell, 62:1143-51 (1990) and humans (Bambara et al., “Reconstitution of Mammalian DNA Replication, “Prog. Nuc. Acid Res.,” 51:93-123 (1995)) have also been characterized in some detail as has herpes virus (Boehmer et al., “Herpes Simplex Virus DNA Replication,” Annu. Rev. Biochem., 66:347-384 (1997)) and vaccinia virus (McDonald et al., “Characterization of a Processive Form of the Vaccinia Virus DNA Polymerase,” Virology, 234:168-175 (1997)). The helicase of E. coli is encoded by the dnaB gene and is called the DnaB-helicase. In phage T4, the helicase is the product of the gene 41, and, in T7, it is the product of gene 4. Generally, the helicase contacts the DNA polymerase in E. coli. This contact is necessary for the helicase to achieve the catalytic efficiency needed to replicate a chromosome (Kim et al., “Coupling of a Replicative Polymerase and Helicase: A tau-DnaB Interaction Mediates Rapid Replication Fork Movement,” Cell, 84:643-650 (1996)). The identity of the helicase that acts at the replication fork in a eukaryotic cellular system is still not firm.
The primase of E. coli (product of the dnaG gene), phage T4 (product of gene 61), and T7 (gene 4) require the presence of their cognate helicase for activity. The primase of eukaryotes, called DNA polymerase alpha, looks and behaves differently. DNA polymerase alpha is composed of 4 subunits. The primase activity is associated with the two smaller subunits, and the largest subunit is the DNA polymerase which extends the product of the priming subunits. DNA polymerase alpha does not need a helicase for priming activity on single strand DNA that is not coated with binding protein.
The chromosomal replicating DNA polymerase of all these systems, prokaryotic and eukaryotic, share the feature that they are processive, meaning they remain continuously associated with the DNA template as they link monomer units (dNTPs) together. This catalytic efficiency can be manifest in vitro by their ability to extend a single primer around a circular ssDNA of over 5,000 nucleotide units in length. Chromosomal DNA polymerases will be referred to here as replicases to distinguish them from DNA polymerases that function in other DNA metabolic processes and are far less processive.
There are three types of replicases known thus far that differ in how they achieve processivity and how their subunits are organized. These will be referred to here as Types I-III. The Type I is exemplified by the phage T5 replicase, which is composed of only one subunit yet is highly processive (Das et al., “Mechanism of Primer-template Dependent Conversion of dNTP-dNMP by T7 DNA Polymerase,” J. Biol. Chem., 255:7149-7154 (1980)). It is possible that the T5 enzyme achieves processivity by having a cavity within it for binding DNA, with a domain of the protein acting as a lid that opens to accept the DNA and closes to trap the DNA inside, thereby keeping the polymerase on DNA during polymerization of dNTPs. Type II is exemplified by the replicases of phage T7, herpes simplex virus, and vaccinia virus. In these systems, the replicase is composed of two subunits, the DNA polymerase and an “accessory protein” which is needed for the polymerase to become highly efficient. It is presumed that the DNA polymerase binds the DNA in a groove and that the accessory protein forms a cap over the groove, trapping the DNA inside for processive action. Type III is exemplified by the replicases of E. coli, phage T4, yeast, and humans in which there are three separate components, a sliding clamp protein, a clamp loader protein complex, and the DNA polymerase. In these systems, the sliding clamp protein is an oligomer in the shape of a ring. The clamp loader is a multiprotein complex which uses ATP to assemble the clamp around DNA. The DNA polymerase then binds the clamp which tethers the polymerase to DNA for high processivity. The replicase of the E. coli system contains a fourth component called tau that acts as a glue to hold two polymerases and one clamp loader together into one structure called Pol III*. In this application, any replicase that uses a minimum of three components (i.e., clamp, clamp loader, and DNA polymerase) will be referred to as either a three component polymerase, a type III enzyme, or a DNA polymerase III-type replicase.
The E. coli replicase is also called DNA polymerase III holoenzyme. The holoenzyme is a single multiprotein particle that contains all the components; it is comprised of ten different proteins. This holoenzyme is suborganized into four functional components called: 1) Pol III core (DNA polymerase); 2) gamma complex or tau/gamma complex (clamp loader); 3) beta subunit (sliding clamp); and 4) tau (glue protein). The DNA polymerase III “core” is a tightly associated complex containing one each of the following three subunits: 1) the alpha subunit is the actual DNA polymerase (129 kDa); 2) the epsilon subunit (28 kDa) contains the proofreading 3′-5′ exonuclease activity; and 3) the theta subunit has an unknown function. The gamma complex is the clamp loader and contains the following subunits: gamma, delta, delta prime, chi and psi (U.S. Pat. No. 5,583,026 to O'Donnell). Tau can substitute for gamma, as can a tau/gamma heterooligomer. The beta subunit is a homodimer and forms the ring shaped sliding clamp. These components associate to form the holoenzyme and the entire holoenzyme can be assembled in vitro from 10 isolated pure subunits (U.S. Pat. No. 5,583,026 to O'Donnell; U.S. Pat. No. 5,668,004 to O'Donnell). The E. coli dnaX gene encodes both tau and gamma. Tau is the product of the full gene. Gamma is the product of the first ⅔ of the gene; it is truncated by an efficient translational frameshift that results in incorporation of one unique residue followed by a stop codon.
The tau subunit, encoded by the same gene that encodes gamma (dnaX), also acts as a glue to hold two cores together with one gamma complex. This subassembly is called DNA polymerase III star (Pol III*). One beta ring interacts with each core in Pol III* to form DNA polymerase III holoenzyme.
During replication, the two cores in the holoenzyme act coordinately to synthesize both strands of DNA in a duplex chromosome. At the replication fork, DNA polymerase III holoenzyme physically interacts with the DnaB helicase through the tau subunit to form a yet larger protein complex termed the “replisome” (Kim et al., “Coupling of a Replicative Polymerase and Helicase: A tau-DnaB Interaction Mediates Rapid Replication Fork Movement,” Cell, 84:643-650 (1996); Yuzhakov et al., “Replisome Assembly Reveals the Basis for Asymmetric Function in Leading and Lagging Strand Replication,” Cell, 86:877-886 (1996)). The primase repeatedly contacts the helicase during replication fork movement to synthesize RNA primers on the lagging strand (Marians, K. J., “Prokaryotic DNA Replication,” Annu. Rev. Biochem., 61:673-719 (1992)).
Intensive subtyping of prokaryotic cells has now lead to a taxonomic classification of prokaryotic cells as eubacteria (true bacteria) to distinguish them from archaebacteria. Within eubacteria are many different subcategories of cells, although they can broadly be subdivided into Gram positive- and Gram negative-like cells. Numerous complete and partial genome sequences of prokaryotes have appeared in the public databases.
In the present invention, new genes from the Gram positive bacteria, Streptococcus pyogenes (e.g., S. pyogenes) and Staphylococcus aureus (e.g., S. aureus) are identified. They are assigned names based on their nearest homology to subunits in the E. coli system. The genes encoding E. coli replication proteins are as follows: alpha (dnaE); epsilon (dnaQ); theta (holE); tau (full length dnaX); gamma (frameshift product of dnaX); delta (holA); delta prime (holB); chi (holC); psi (holD); beta (dnaN); DnaB helicase (dnaB); and primase (dnaG).
Study of the organisms for which a complete genome sequence is available reveals that no organism has identifiable homologues to all the subunits of the E. coli three component polymerase, Pol III holoenzyme (see Table 1 below). All other organisms lack the 0 subunit (holE), and all except one lack genes encoding the χ and ψ subunits (holC and holD, respectively) as judged by sequence comparison searches. Further, the α and ε subunits are fused into one large a subunit in some organisms (e.g., Gram positive cells) as detailed in (Sanjanwala et al., “DNA Polymerase III Gene of Bacillus subtilis,” Proc. Natl. Acad. Sci. USA, 86:4421-4424 (1989)). Although all organisms have homologues to τ, β, δ′ and SSB, the δ subunit has diverged significantly (either not recognized or nearly not recognized by gene searching programs), perhaps even to the point where it is no longer involved in DNA replication. The DnaX product also would appear to lack frameshift signals in most organisms. This predicts only one protein (tau) will be produced from this gene, instead of two as in E. coli. Indeed, this has been shown to be true for the Staphylococcus aureus DnaX (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference). Finally, genetic study of Bacillus subtilis identified two genes that do not have counterparts in E. coli (dnaB, not the helicase, and dnaH) as well as one other gene, dnaI, that is only very distantly related to E. coli dnaC (Karamata et al., “Isolation and Genetic Analysis of Temperature-Sensitive Mutants of B. subtilis Defense in DNA Synthesis,” Molec. Gen. Genet., 108:277-287 (1970); Braund et al., “Nucleotide Sequence of the Bacillus subtilis dnaD Gene,” Microb., 141:321-322 (1995); Hoshino et al., “Nucleotide Sequence of Bacillus subtilis dnaB: A Gene Essential for DNA Replication Initiation and Membrance Attachment,” Proc. Natl. Acad. Sci. USA,” 84:653-657 (1987)). Keeping in mind the apparently random, or at least unpredictable process of evolution, it is possible that these apparently new genes perform novel functions that may result in a new type of polymerase for chromosomal replication. Thus, it seems possible that new proteins may have evolved to take the place of χ, ψ, θ, the frameshift product of DnaX, and possibly δ in other eubacteria. These considerations indicate that the three component polymerase of different eubacteria may have different structures. That this may be so would not be surprising as different bacteria are often less related evolutionarily than plants are to humans. For example, the split between Gram positive and Gram negative bacteria occurred about 1.2 billion years ago. This distant split makes Gram positive cells an attractive source to examine how different other eubacterial three component polymerases are from the E. coli Pol III holoenzyme.
TABLE 1Organism (Order)χφθεαβdnaXδ′δEscherichia coli+++++++++ProteobacteriaHaemophilus influenzae++−++++++ProteobacteriaMycoplasma genitalium−−−−+++++(weak)FirmicutesSynichisystis sp.−−−−+++++(weak)CyanobacteriaBacillus subtilis−−−−+++++(weak)FirmicutesBorrelia burgdorferi−−−−+++++(weak)SpirochaetalesAquifex aeolicus−−−++++++(weak)AquificalesMycobacterium tuberculosis−−−++++++(weak)Firmicutes & ActinobacteriaTreponema pallidum−−−++++++(weak)SpirochaetalesChlamydia trachomatis−−−++++++(weak)ChlamydialesRickettsia prowazekii−−−++++++(weak)ProteobacteriaHelicobacter pylori−−−++++++(weak)ProteobacteriaThermatoga maritima−−−−+++++(weak)Thermotogales
The goal of this invention is to learn how to form a functional three component polymerase from an organism that is highly divergent from E. coli and whether it is as rapid and processive as the E. coli Pol III holoenzyme. Namely, from bacteria lacking χ, ψ, or θ, or having a widely divergent δ subunit, or having only one DnaX product, or an α subunit that encompasses both α and ε activities. All eubacteria for which the entire genome has been sequenced have at least one of these differences from E. coli. Many Gram negative bacteria have one or more of these differences (e.g., Haemophilus influenzae and Aquifex aeolicus). Bacteria of the Gram positive class have all of these different features. Because of the distant evolutionary split between Gram positive and Gram negative bacteria, their mechanisms of replication may have diverged significantly as well. Indeed, purification of the replication polymerase from B. subtilis, a Gram positive cell, gives only a single subunit polymerase (Barnes et al., “Purification of DNA Polymerase III of Gram-Positive Bacteria,” Methods Enzy. 262:35-42 (1995); Barnes et al., “Antibody to B. subtilis DNA Polymerase III: Use in Enzyme Purification and Examination of Homology Among Replication-specific DNA Polymerases,” Nucl. Acids Res., 6:1203-209 (1979); Barnes et al., “DNA Polymerase III of Mycoplasma pulmonis: Isolation and Characterization of the Enzyme and its Structural Gene, polC,” Mol. Microb., 13:843-854, (1994); Low et al., “Purification and Characterization of DNA Polymerase III from Bacillus subtilis,” J. Biol. Chem., 251:1311-1325 (1976)) instead of a 10 subunit assembly containing the three components of a rapidly processive machine as discussed above for Pol III holoenzyme from E. coli. This finding suggests a different structural organization of the replicase and possibly different functional characteristics as well.
Although there are many studies of replication mechanisms in eukaryotes and, specifically, the Gram negative bacterium E. coli and its bacteriophages, there is very little information about how Gram positive organisms replicate. The Gram positive class of bacteria includes some of the worst human pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, and Mycobacterium tuberculosis (Youmans et al., The Biological and Clinical Basis of Infectious Disease (1985)). Until this invention, the best characterized Gram positive organism for chromosomal DNA synthesis was Bacillus subtilis. Fractionation of B. subtilis has identified three DNA polymerases. (Gass et al., “Further Genetic and Enzymological Characterization of the Three Bacillus subtilis Deoxyribonucleic Acid Polymerases,” J. Biol. Chem., 248:7688-7700 (1973); Ganesan et al., “DNA Replication in a Polymerase I Deficient Mutant and the Identification of DNA Polymerases II and III in Bacillus subtilis,” Biochem. Biophys. Res. Commun., 50:155-163 (1973)). These polymerases are thought to be analogous to the three DNA polymerases of E. coli (DNA polymerases I, II, and III). Studies in B. subtilis have identified a polymerase that appears to be involved in chromosome replication and is termed Pol III (Ott et al., “Cloning and Characterization of the polC Region of Bacillus subtilis,” J. Bacteriol., 165:951-957 (1986); Barnes et al., “Localization of the Exonuclease and Polymerase Domains of Bacillus subtilis DNA Polymerase III,” Gene, 111:43-49 (1992); Barnes et al., “The 3′-5′ Exonuclease Site of DNA Polymerase III From Gram-positive Bacteria: Definition of a Novel Motif Structure,” Gene” 165:45-50 (1995) or Barnes et al., “Purification of DNA Polymerase III of Gram-positive Bacteria,” Methods in Enzy., 262:35-42 (1995)). The B. subtilis Pol III (encoded by polC) is larger (about 165 kDa) than the E. coli alpha subunit (about 129 kDa) and exhibits 3′-5′ exonuclease activity. The polC gene encoding this Pol III shows weak homology to the genes encoding E. coli alpha and the E. coli epsilon subunit. Hence, this long form of the B. subtilis Pol III (herein referred to as α-large or Pol III-L) essentially comprises both the alpha and epsilon subunits of the E. coli core polymerase. The S. aureus α-large has also been sequenced, expressed in E. coli, and purified; it contains DNA polymerase and 3′-5′ exonuclease activity (Pacitti et al., “Characterization and Overexpression of the Gene Encoding Staphylococcus aureus DNA Polymerase III,” Gene, 165:51-56 (1995)). Although α-large is essential to cell growth (Clements et al., “Inhibition of Bacillus subtilis Deoxyribonucleic Acid Polymerase III by Phenylhydrazinopyrimidines: Demonstration of a Drug-induced Deoxyribonucleic Acid-Enzyme Complex,” J. Biol. Chem., 250:522-526 (1975); Cozzarelli et al., “Mutational Alteraction of Bacillus subtilis DNA Polymerase III to Hydroxyphenylazopyrimidine Resistance: Polymerase III is Necessary for DNA Replication,” Biochem. And Biophy. Res. Commun., 51:151-157 (1973); Low et al., “Mechanism of Inhibition of Bacillus subtilis DNA Polymerase III by the Arylhydrazinopyrimidine Antimicrobial Agents,” Proc. Natl. Acad. Sci. USA, 71:2973-2977 (1974)), there could still be another DNA polymerase(s) that is essential to the cell, such as occurs in yeast (Morrison et al., “A Third Essential DNA Polymerase in S. cerevisiae,” Cell, 62:1143-1151 (1990)).
Purification of α-large from B. subtilis results in only this single protein without associated proteins (Barnes et al., “Localization of the Exonuclease and Polymerase Domains of Bacillus subtilis DNA Polymerase III,” Gene, 111:43-49 (1992); Barnes et al., “The 3′-5′ Exonuclease Site of DNA Polymerase III From Gram-positive Bacteria: Definition of a Novel Motif Structure,” Gene” 165:45-50 (1995) or Barnes et al., “Purification of DNA Polymerase III of Gram-positive Bacteria,” Methods in Enzymol., 262:35-42 (1995)). Hence, it is possible that α-large is a member of the Type I replicase (like T5) in which it is processive on its own without accessory proteins. B. subtilis and S. aureus also have a gene encoding a protein that has approximately 30% homology to the beta subunit of E. coli; however, the protein product has not been purified or characterized (Alonso et al., “Nucleotide Sequence of the recF Gene Cluster From Staphylococcus aureus and Complementation Analysis in Bacillus subtilis recF Mutants,” Mol. Gen. Genet., 246:680-686 (1995); Alonso et al., “Nucleotide Sequence of the recF Gene Cluster From Staphylococcus aureus and Complementation Analysis in Bacillus subtilis recF Mutants,” Mol. Gen. Genet., 248:635-636 (1995)). Whether this beta subunit has a function in replication, a ring shape, or functions as a sliding clamp was not known until recently. It was also not known whether it is functional with α-large. Recently, it was shown that S. aureus β is functional as a ring, and that it also functions with α-large (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference). Further, a fourth DNA polymerase was identified with greater homology to E. coli a than α-large. This polymerase, called herein α-small, is shorter than α-large and lacks the domain homologous to epsilon. This polymerase also functions with the β ring, indicating that it may participate in chromosome replication. Indeed, a recent report indicates that α-small is essential for replication in Streptomyces coelicolor A3(2) (Flett et al., “A Gram-negative type” DNA Polymerase III is Essential for Replication of the Linear Chromosome of Streptomyces Coelicolor A3(2),” Mol. Micro., 31:949-958, (1999)).
As described earlier, purification of the replicase from the Gram positive B. subtilis gives only a single subunit Pol II, instead of a multicomponent complex. Also, S. aureus dnaX has been shown to encode only one subunit (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference). Moreover, S. aureus and B. subtilis lack homologues to χ, ψ, θ, and the δ subunit is only weakly homologous to 8 of E. coli (only 28%). Further, they lack a homologue to dnaQ encoding E. Instead, they contain this activity (3′-5′ exonuclease) in the polC gene product which provides the α-large form of α. The ε subunit is needed for high speed and processivity of the E. coli Pol III holoenzyme; the α subunit alone is much less rapid and processive with the β ring compared to the presence of both α and β (Studwell et al., “Processive Replication is Contingent on the Exonuclease Subunit of DNA Polymerase III Holoenzyme,” J. Biol Chem, 265: 1171-1178 (1990)).
Studies using the E. coli β ring (and γ complex) show they confer onto S. aureus a quite efficient synthesis (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference), but the efficiency is not equal to that of E. coli αε with β (and γ complex). This may be due to use of the heterologous combination of an α subunit from one organism (S. aureus) with the β clamp from another (E. coli.). However, it is also possible that S. aureus α simply does not function with a β clamp to produce speed and processivity comparable to the E. coli polymerase. Also, as described earlier, the α-large subunit of B. subtilis purifies as a single subunit, rather than associated with accessory subunits assembled into the three components of a rapid, processive machine (i.e., like E. coli Pol III holoenzyme). The lack of two DnaX products, lack of a multicomponent structure, and lack of gene homologues encoding several subunits of the three component, Pol III, of E. coli brings into question whether other types of bacteria, such as Gram positive cells, even have an enzyme with similar structure or comparable speed and processivity to that found in the Gram negative E. coli. 
The lack of gene homologues encoding several subunits of the E. coli three component polymerase creates uncertainties with respect to reconstructing a rapid and processive polymerase from a Gram positive cell that has characteristics like the Pol III system of E. coli. 
The γ and δ′ proteins are homologous to one another, encoding C-shape proteins Long et al., “DNA Polymerase III Accessory Proteins,” J. Biol. Chem., 268:11758-11765, (1993); Guenther et al., “Crystal Structure of the 8′ Subunit of the Clamp-loader Complex of E. coli DNA Polymerase III,” Cell, 91:335-345 (1997)). The clamp loaders of yeast and humans are composed of five proteins, all of which are homologous to one another and to γ and δ′ (Cullman et al., “Characterization of the Five Replication Factor C Genes of Saccharomyces Cerevisiae,” Mol. Cell. Biol., 15:4661-4671 (1995)). This provides evidence that a clamp loader can be composed entirely of C-shape proteins. Perhaps the Gram positive DnaX-protein (hereafter referred to as τ) and δ′ are sufficient to provide function as a clamp loader. Indeed, the clamp loader of T4 phage is composed of only two different proteins, gp44/62 complex (Young et al., “Structure and Function of the Bacteriophage T4 DNA Polymerase Holoenzyme,” Biochem., 31:8675-8690 (1992)). This idea is also supported by the presence of only two RFC genes in archaebacteria, suggesting that they may utilize two C-shaped proteins for clamp loading, in contrast to yeast and humans that use five. With this consideration in mind, genes were identified and isolated and the τ protein (encoded by dnaX) and 6′ (encoded by holB) of another Gram positive organism, Streptococcus pyogenes, were expressed and purified. As was observed in S. aureus, S. pyogenes dnaX produces only a single polypeptide. The β, encoded by dnaN of S. pyogenes, was also identified, expressed, and purified, as were the α-large subunit encoded by polC and the SSB encoded by the ssb gene. These proteins were studied for interactions and characterized for their effect on α-large. However, the hypothesis was incorrect as T and δ′ did not form a τδ′ complex, nor did they assemble β onto DNA or provide stimulation of a when using β on primed and SSB coated M13 mp18 ssDNA.
In light of the inability of S. pyogenes τ protein and δ′ to function as a clamp loader, it seemed reasonable to expect that one or more other proteins are needed. The fact that E. coli has some replicase subunits that other bacteria do not, suggests that other bacteria may have some replicase subunits that E. coli does not. Indeed, genetic studies of Bacillus subtilis demonstrates that it has three genes needed for replication that E. coli does not have. Two of these novel genes, called dnaB (not the same as E. coli dnaB encoding the helicase) and dnaH, have no significant homology to genes in the E. coli genome database (Bruand et al., “Nucleotide Sequence of the Bacillus subtilis dnaD gene,” Microbiol., 141:321-322 (1995); Hoshino et al., “Nucleotide Sequence of Bacillus subtilis dnaB: A gene Essential for DNA replication Initiation and Membrane Attachment,” Proc. Natl. Acad. Sci. USA, 84:653-657 (1987)). Further, dnaI of B. subtilis is important for replication and has, at best, a very limited homology to E. coli dnaC (Karamata et al., “Isolation and Genetic Analysis of Temperature-Sensitive Mutants of B. subtilis Defective in DNA synthesis,” Molec. Gen. Genetics, 108:277-287 (1970)). Perhaps one or more of these genes encode the proteins(s) necessary to provide clamp loading activity when combined with τ and δ′, or to couple with a to provide it with speed and/or processivity as the E. coli epsilon does. The S. pyogenes homologues of B. subtilis dnaI, dnaH, and dnaB were identified, cloned, and the encoded proteins were expressed and purified. However, these proteins failed to provide activity alone or in combinations with S. pyogenes τ and δ′ in loading S. pyogenes onto DNA, or in stimulating S. pyogenes α-large in combination with β, τ, and δ′ on SSB coated primed M13 mp18 ssDNA.
Weak homology exists for the holA gene among prokaryotes. This weak homologue of holA was identified in S. pyogenes and, then, it was cloned, expressed, and the putative δ was purified. The putative δ formed an isolatable complex with τ and δ′. In fact, the τδδ′ complex loaded S. pyogenes β onto DNA, and it stimulated S. pyogenes α-large in a β dependent reaction on primed SSB coated M13 mp18 ssDNA. Hence, this protein was the only missing component necessary to provide clamp loading activity. Further, a mixture of a with τδδ′, followed by ion exchange chromatography on MonoQ, indicated formation of an ατδδ′ complex. Consistent with this, τ appeared to bind a in gel filtration analysis.
Whether the S. pyogenes three component polymerase can synthesize DNA in as rapid and processive of a fashion as the E. coli Pol III holoenzyme three component polymerase is very difficult to predict, because no other DNA polymerase known to date catalyzes synthesis at the rate or processivity of the E. coli three component polymerase. For example, the three component T4 phage polymerase travels about 400 nucleotides/s, the yeast DNA polymerase delta three component polymerase travels about 120 nucleotides/s, and the human DNA polymerase delta three component enzyme appears slower and less processive than the yeast enzyme.
The standard test for these speed and processivity characteristics is examination of a time course in extension of a primer on a very long template, such as around the 7.2 kb M13mp18 ssDNA genome coated with SSB and primed with a synthetic DNA oligonucleotide. The results of experiments of this type demonstrate that the three component S. pyogenes polymerase is indeed extremely rapid in synthesis. Surprisingly, it is just as fast as the E. coli enzyme. Extension proceeds at about 700-800 nucleotides per second, completing the entire template in about 9 seconds. The enzyme was fully processive throughout replication of the M13mp18 genome, as could be determined from the fact that some templates were not extended at all, while others were extended to completion. If the enzyme had not been processive during the entire replication reaction, then when it comes off one partially extended DNA genome it would have reassociated with the unextended DNA that remained and partially replicated it as well (and so on until the entire population of DNA became fully replicated). This did not happen. Instead, the reaction showed a mixture of completely replicated templates and templates that were still untouched starting material. This indicates that the enzyme stays with a template until it completes it before it cycles over to replicate another one (i.e., it is highly processive). Each of the five proteins, α, τ, δ, δ′ and β, are needed to obtain this rapid and processive DNA synthesis.
This invention has provided an intellectual template by which the clamp loader component of these three component polymerases can be obtained from any eubacterial prokaryotic cell and how to use it with the other components to produce a rapid and processive polymerase. All prokaryotes in the eubacterial kingdom that have been sequenced to date contain homologues of these genes. As the process of lateral gene transfer appears to be a major force in evolution, it would appear that relatedness of enzymes and enzyme machines is best judged by comparisons of their genes and proteins rather than by phylogeny of which bacteria they are in (Doolittle et al., “Archaeal Genomics: Do Archaea have a Mixed Heritage?,” Curr. Biol., 8:R209—R211 (1998)). As pointed out earlier in this application, most bacteria have genetic characteristics of replication genes/proteins of S. pyogenes rather than that of E. coli (i.e., no genes encoding χ, ψ, or θ, only a weak homolog to δ, or a dnaX gene encoding only a single protein).
The dnaX gene encoding τ and γ in E. coli encodes only one protein in some organisms, but, as this application shows, it is still functional in forming a protein complex capable of rapid and processive DNA synthesis. In addition, this application shows that the delta subunit, which is only weakly homologous among different prokaryotic organisms, is an essential functional subunit of the three component polymerase (instead of having diverged so as to fulfill an entirely different function in some other intracellular process). As mentioned earlier, several genes encoding subunits of the E. coli clamp loader (γ complex; γ, δ,δ′,χ,ψ) are not obviously present in other prokaryotes (holC and holD encoding χ and ψ). Hence, one may anticipate that other genes may have evolved to encode new subunits that replace these, and that these new subunits may have been essential to the activity of the clamp loader. For example, they may have either taken over some of the functionality of another subunit, or structurally (e.g., the physical presence of a subunit could be needed for one subunit to assume its proper and active conformation, or for one or more of the subunits to form a complex together to yield the multisubunit clamp loader assembly). In addition, this application shows that the α subunit (polC gene product) is sufficient for rapid and processive synthesis with the other two components (i.e., E. coli requires ε submit to bind to α for rapid and processive synthesis of a with the β clamp). Finally, this application shows that the S. pyogenes three component polymerase synthesizes DNA as fast as the E. coli Pol III three component polymerase. Up to this point, the E. coli Pol III three component polymerase was over twice the speed of the T4 enzyme and over 5 times the speed of others. Hence, it was possible that E. coli may have been unique among prokaryotes in having a polymerase that achieves such speed. This invention shows that this is not the case. Instead, this speed in polymerization generalizes to the Gram positive prokaryotic three component DNA polymerases. It may be presumed, now that two examples of three component polymerases in widely divergent bacteria share the characteristics of rapid, processive synthesis, that the three component polymerase of other eubacteria will also be rapid and processive.
These rapid and processive three component DNA polymerases can be applied to several important uses. DNA polymerases currently in use for DNA sequencing and DNA amplification use enzymes that are much slower and thus could be improved upon. This is especially true of amplification as the three component polymerase is capable of speed and high processivity making possible amplification of very long (tens of Kb to Mb) lengths of DNA in a time efficient manner. These three component polymerases also function in conjunction with a replicative helicase (DnaB) and, thus, are capable of amplification at ambient temperature using the helicase to melt the DNA duplex. This property could be useful in amplification reaction procedures such as in polymerase chain reaction (PCR) methodology. Finally, these three component polymerases and their associated helicase (DnaB) and primase (DnaG) are attractive targets for antibiotics due to their essential and central role in cell viability.
This application provides a three component polymerase from two human pathogens in the Gram positive class. It makes possible the production of this three component polymerase from other bacteria of the Gram positive type (e.g., Streptococci, Staphylococci, Mycoplasma) and other types of bacteria lacking χ, ψ, or θ, those having only one protein produced by their dnaX gene such as obligate intracellular parasites, Mycoplasmas (possibly evolved from Gram positives), Cyanobacteria (Synechocystis), Spirochaetes such as Borrelia and Treponemia and Chlamydia, and distant relatives of E. coli in the Gram negative class (e.g., Rickettsia and Helicobacter). These three component polymerases are useful in manipulation of nucleic acids for research and diagnostic purposes (e.g., sequencing and amplification methods) and for screening chemicals for antibiotic activity (useful in human or animal therapy and agriculture such as animal feed supplements). There are several assays described previously in U.S. patent application Ser. No. 09/235,245 to O'Donnell et al., which is hereby incorporated by reference, that use these three component polymerases (or subassemblies), as well as the DnaB and DnaG homologues, either alone or in various combinations, for the purpose of screening chemicals, such as chemical libraries, for inhibitor activity. Such inhibitors can be developed further (usually by chemical manipulation and alteration) into lead compounds and then into full fledged pharmaceuticals.
There remains a need to understand the molecular details of the process of DNA replication in other cells that are quite different from E. coli, such as in Gram positive cells. It is possible that a more detailed understanding of replication proteins will lead to discovery of new antibiotics. Therefore, a deeper understanding of replication proteins of Gram positive bacteria is especially important given the emergence of drug resistant strains of these organisms. For example, Staphylococcus aureus has successfully mutated to become resistant to all common antibiotics.
The “target” protein(s) of an antibiotic drug is generally involved in a critical cell function, such that blocking its action with a drug causes the pathogenic cell to die or no longer proliferate. Current antibiotics are directed to very few targets. These include membrane synthesis proteins (e.g., vancomycin, penicillin, and its derivatives such as ampicillin, amoxicillin, and cephalosporin), the ribosome machinery (e.g., tetracycline, chloramphenicol, azithromycin, and the aminoglycosides such as kanamycin, neomycin, gentamicin, streptomycin), RNA polymerase (e.g., rifampimycin), and DNA topoisomerases (e.g., novobiocin, quinolones, and fluoroquinolones). The DNA replication apparatus is a crucial life process and, thus, the proteins involved in this process are good targets for antibiotics.
A powerful approach to discovery of a new drug is to obtain a target protein, characterize it, and develop in vitro assays of its cellular function. Large chemical libraries can then be screened in the functional assays to identify compounds that inhibit the target protein. These candidate pharmaceuticals can then be chemically modified to optimize their potency, breadth of antibiotic spectrum, non-toxicity, performance in animal models and, finally, clinical trials. The screening of large chemical libraries requires a plentiful source of the target protein. An abundant supply of protein generally requires overproduction techniques using the gene encoding the protein. This is especially true for replication proteins as they are present in low abundance in the cell.
Selective and robust assays are needed to screen reliably a large chemical library. The assay should be insensitive to most chemicals in the concentration range normally used in the drug discovery process. These assays should also be selective and not show inhibition by antibiotics known to target proteins in processes outside of replication.
The present invention is directed to overcoming these deficiencies in the art.